CN103604783A - Method for reversible detection on hypochlorite and hydrogen sulfide - Google Patents
Method for reversible detection on hypochlorite and hydrogen sulfide Download PDFInfo
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- CN103604783A CN103604783A CN201310539154.3A CN201310539154A CN103604783A CN 103604783 A CN103604783 A CN 103604783A CN 201310539154 A CN201310539154 A CN 201310539154A CN 103604783 A CN103604783 A CN 103604783A
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- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 title claims abstract description 93
- 229910000037 hydrogen sulfide Inorganic materials 0.000 title claims abstract description 93
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000002441 reversible effect Effects 0.000 title claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 154
- 150000004696 coordination complex Chemical class 0.000 claims abstract description 6
- 238000004020 luminiscence type Methods 0.000 claims description 66
- 239000007853 buffer solution Substances 0.000 claims description 23
- 230000005284 excitation Effects 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 9
- -1 halide ion Chemical group 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 229910052762 osmium Inorganic materials 0.000 claims description 5
- 229910052707 ruthenium Inorganic materials 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 125000004890 (C1-C6) alkylamino group Chemical group 0.000 claims description 2
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 claims description 2
- 229910020366 ClO 4 Inorganic materials 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims description 2
- 230000005477 standard model Effects 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims 1
- 230000033116 oxidation-reduction process Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 101
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 101
- 229910019142 PO4 Inorganic materials 0.000 description 31
- 239000008366 buffered solution Substances 0.000 description 31
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 31
- 239000010452 phosphate Substances 0.000 description 31
- 230000004044 response Effects 0.000 description 30
- 238000003384 imaging method Methods 0.000 description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
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- 239000001301 oxygen Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 5
- 229950000688 phenothiazine Drugs 0.000 description 5
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- 0 CC=C(C=C(CCC(C)=C)c1cc(*)ccn1)I Chemical compound CC=C(C=C(CCC(C)=C)c1cc(*)ccn1)I 0.000 description 3
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- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 239000007850 fluorescent dye Substances 0.000 description 2
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- 150000002894 organic compounds Chemical class 0.000 description 2
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 2
- 125000001484 phenothiazinyl group Chemical group C1(=CC=CC=2SC3=CC=CC=C3NC12)* 0.000 description 2
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 2
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
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- 241000370738 Chlorion Species 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a method for reversible detection on hypochlorite and hydrogen sulfide, and in particularly relates to a light emission detection method which is high in selectivity, high in sensitivity, and capable of reversibly detecting hypochlorite, hydrogen sulfide and oxidation reduction circulation of hypochlorite and hydrogen sulfide, and an application of the method. The method for reversible detection on hypochlorite and hydrogen sulfide is a method which is used for detecting by utilizing light emitting probe molecules, wherein the light emitting probe molecules are a metal complex (L1L2)M-L3Y3 having the structure as shown in a general formula I. The metal complex can be used as the light emitting probe molecules which are high in selectivity, high in sensitivity, and capable of detecting hypochlorite, hydrogen sulfide and oxidation reduction circulation of hypochlorite and hydrogen sulfide in a circulation and reversion manner. By adopting the light emitting probe molecules, circulatory and reversible detection on hypochlorite, hydrogen sulfide and oxidation reduction process of hypochlorite and hydrogen sulfide are achieved.
Description
Technical field
The present invention relates to a kind of method of reversible detection hypochlorite and sulfuretted hydrogen, be specifically related to luminous detection method and the application thereof of a class selectivity, highly sensitive, reversible detection hypochlorite and sulfuretted hydrogen and redox cycle thereof.
Background technology
Hypochlorite ion is widely used in daily life, such as home disinfecting agent and bleaching agent etc.Meanwhile, hypochlorite ion is also a kind of important activity oxygen species in biosome.Under the catalysis of myeloperoxidase (MPO), hydrogen peroxide and chlorion react, and produce hypochlorous acid.Under normal circumstances, in biosome, hypochlorite ion's amount maintains in a tension metrics level, but after in body, the amount of myeloperoxidase morphs, in biosome, hypochlorite ion's amount will be unbalance, thereby brings out the generation of various diseases.For example: angiocardiopathy (S.Sugiyama, Y.Okada, G.K.Sukhova, R.Virmani, J.W.Heinecke, P.Libby, Am.J.Pathol.2001,158,879 – 891; S.Sugiyama, K.Kugiyama, M.Aikawa, S.Nakamura, H.Ogawa, P.Libby, Arterioscler.Thromb.Vasc.Biol.2004,24,1309 – 1314), neuronal degeneration (Y.W.Yap, M.Whiteman, N.S.Cheung, Cell.Signalling2007,19,219 – 228.), arthritis (M.J.Steinbeck, L.J.Nesti, P.F.Sharkey, J.Parvizi, J.Orthop.Res.2007,25,1128 – 1135.), even may bring out (S.A.Weitzman, L.I.Gordon, the Blood1990 such as cancer, 76,655 – 663.).Therefore the method for finding quick, sensitive, single-minded detection hypochlorite ion, has caused great attention in association areas such as medical science, biology, biological chemistry and environmental chemistries in recent years.
In biosome and cell, there are various signal pathways, because gaseous signal molecule has, can produce continuously, propagate the features such as rapid, quick disperse, caused people's extensive concern.Since the nineties in 20th century, people recognize that sulfuretted hydrogen is a kind of novel endogenous gaseous signal molecule (H.Kimura, the Y.Nagai being present in biosome gradually, K.Umemura and Y.Kimura, Antioxid.Redox Signaling, 2005,7,795; B.Predmore, D.Lefer and G.Gojon, Antioxid.Redox Signaling, 2012,17,119.).This gas can, by the catalytic action of enzyme, produce in a lot of organs in vivo.Although the metabolic pathway of sulfuretted hydrogen is still not very clear, increasing result of study confirmation in recent years, H
2s is at SH, chronic obstructive emphysema, septicopyemia or hemorrhagic shock, Alzheimer disease, in the production process of the various diseases such as mucosal lesion and cirrhosis, bringing into play very important Pathological Physiology effect (Geng B.Yang J.Qi Y H2S generated by heart in rat and effects on cardiac function2004 (02) .Geng B.Chang L.Pan CS Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol2004 (03).Therefore in the urgent need to search out can Sensitive Detection body in the detection method of hydrogen sulfide content, realize the effective monitoring to sulfuretted hydrogen.
At present, about detecting hypochlorite (Lin Yuan, Weiying Lin, Jizeng Song, Yueting Yang., Chem.Commun.2011,47,12691 – 12693; Zhangrong Lou, Peng Li, Qiang Pan, Keli Han., Chem.Commun., 2013,49; 2445-2447) and sulfuretted hydrogen (H.Peng, Y.Cheng, C.Dai, A.King, B.Predmore, D.Lefer; B.Wang, Angew.Chem., Int.Ed., 2011,50,9672; Y.Qian, J.Karpus, O.Kabil, S.Zhang, H.Zhu, R.Banerjee, J.Zhao and C.He, Nat.Commun., 2012,2,495) fluorescence probe existing relevant report respectively all.In view of hypochlorite and sulfuretted hydrogen can coexist in vivo, research has reversible redox activity, can either be oxidized by hypochlorite, the fluorescent probe molecule of hydrogen reduction can cure again, thereby realize the selectivity of hypochlorite and sulfuretted hydrogen and redox cycle thereof, highly sensitive, circulation, reversible detection, development of new fluorescent probe molecule and application process thereof, have very important theory and practical value.
Sum up finding, is all to adopt organic compound as fluorescence probe in existing report, and that the shortcoming of most of organic compound is exactly oxidation-reduction stability is poor, cannot realize reversible, cycle detection.
Summary of the invention
A kind of method that the object of this invention is to provide reversible detection hypochlorite and sulfuretted hydrogen.The present invention utilizes tris (bipyridine) ruthenium metal complex to have good light stability, Stokes shift is large, luminescent quantum productive rate is moderate, and this metal complexes has the feature of good reversible redox activity, use it for the selectivity of hypochlorite and sulfuretted hydrogen and redox cycle thereof, highly sensitive, circulation, reversible detection.
A method for reversible detection hypochlorite and sulfuretted hydrogen, comprises the steps:
1. known hypochlorite concentration standard sample and luminescence probe molecule are dissolved in buffer solution, measure system luminous intensity, set up hypochlorite concentration and luminescence probe mulecular luminescence strength relationship;
2. known concentration of hydrogen sulfide standard model is added step 1. in the oxidized luminescence probe molecule buffer solution of gained, measure system luminous intensity, set up concentration of hydrogen sulfide and luminescence probe mulecular luminescence strength relationship;
3. hypochlorite sample to be measured and luminescence probe molecule are dissolved in buffer solution, measure system luminous intensity, according to step 1. gained hypochlorite concentration and system luminous intensity relationship, determine the concentration of hypochlorite in hypochlorite sample to be measured;
4. sulfuretted hydrogen sample to be measured is added step 3. in gained solution, measure system luminous intensity, according to step 2. gained concentration of hydrogen sulfide and system luminous intensity relationship, determine the concentration of sulfuretted hydrogen in sulfuretted hydrogen sample to be measured;
Wherein, described luminescence probe molecule is the metal complex with general formula I structure:
(L
1L
2)M-L
3Y
3
I
In formula I, M is Ru or Os;
Y is halide ion, ClO
4 -, BF
4 -, PF
6 -or OTs
-;
L
1and L
2be selected from independently of one another following part:
Wherein, R
1and R
2be selected from independently of one another H, C
1-18alkyl, CHO, COOH, NH
2, C
1-6alkyl amino, OH, SH, C
1-6alkoxy, C
1-6amide group, C
1-6alkyl replaces or unsubstituted benzyl, halogen or C
1-6haloalkyl.
L
3be selected from following part:
Wherein, R
3for H, CH
3or R
4;
R
4be selected from and there is general formula PTZ
1, PTZ
2or PTZ
3group,
In formula: n=1～18, m=0～18
In said method, preferred described buffer solution is phosphoric acid, boric acid or Tris-HCl buffer solution;
In said method, the concentration of preferred described buffer solution is 0.01～1M;
In said method, the pH=7.4 of preferred buffer solution.
In said method, in the fluoroscopic examination instrument that the mensuration of luminescence probe mulecular luminescence intensity can be buied in any business, carry out ，Ru U.S. Pekin-Elmer LS55 type fluorescence detector.
In said method, preferred described luminescence probe mulecular luminescence intensity is determined as follows: solution is carried out to fluorescence spectrum scanning, and excitation wavelength is 450nm, and emission wavelength sweep limit is 470nm～700nm.
In said method, the concentration of preferred described luminescence probe molecule in buffer solution is 1 * 10
-5mol/L.
L of the present invention
1, L
2and L
3two N atoms in part and M form coordination bond.
The preferred described L of method of reversible detection hypochlorite of the present invention and sulfuretted hydrogen
1and L
2be selected from independently of one another following part:
Wherein, R
1and R
2be selected from independently of one another H and C
1-6alkyl.
The preferred described L of method of reversible detection hypochlorite of the present invention and sulfuretted hydrogen
3be selected from following part:
Wherein, R
3for H or CH
3; R
4be selected from and there is general formula PTZ
2or PTZ
3group, in formula, m=0, n=2～6.
The method of reversible detection hypochlorite of the present invention and sulfuretted hydrogen most preferably described luminescence probe molecule is selected from a kind of in following metal complex:
Luminescence probe molecule of the present invention is preferably by the disclosed method preparation of following document: Chinese invention patent, publication number 101531683A, the interior bipyridyl ruthenium/osmium ECL label with phenothiazine power supplying groups of molecule.
Take Ru-PTZ as example, and the principle of work of luminescence probe molecule of the present invention is:
S atom in luminescence probe molecule on phenothiazine group can be by ClO
-be oxidized to S=O, make the luminous remarkable enhancing of system.And H
2s can reduce S=O, makes probe molecule get back to original state, and the luminous of system is quenched.As long as with phenothiazine group, just can carry out above-mentioned reversible redox circular response in probe molecule, the detection of the variation realization by system luminous intensity to hypochlorite and sulfuretted hydrogen.
The invention has the beneficial effects as follows: the bipyridyl ruthenium/osmium metal complex with phenothiazine power supplying groups in luminescence probe molecule of the present invention has redox reversible.Such complex can selectivity, highly sensitive detection hypochlorite, follows significant luminous enhancing in process.While reaching balance, whole system can also produce selectivity, highly sensitive response to sulfuretted hydrogen, makes luminous cancellation and returns to original state.Said process can constantly move in circles, so this metal complexes can be as the luminescence probe molecule of a class selectivity, highly sensitive, circulation, reversible detection hypochlorite and sulfuretted hydrogen and redox cycle process thereof.Utilize such luminescence probe molecule can realize the circulation of hypochlorite and sulfuretted hydrogen and oxidation-reduction process thereof, reversible detection.
Accompanying drawing explanation
Fig. 1 is embodiment 1,1 * 10
-5the Ru-PTZ of mol/L respectively with (1 * 10 of same concentrations
-4mol/L) sodium hypochlorite, sodium molybdate, H
2o
2,
.oH, in the 0.1mol/L of pH=7.4 phosphate buffered solution, utilizes probe that fluorescence detector the records response condition to each active oxygen species.
Wherein horizontal ordinate is the response time; Ordinate is the luminous intensity that adds the luminous intensity of probe after different active oxygen species to deduct to add probe before different active oxygen species, and the luminous intensity of mixed solution deducts the luminous intensity of probe blank solution.
Fig. 2 is embodiment 2,1 * 10
-5the Ru-PTZ of mol/L and sodium hypochlorite, in the 0.1mol/L of pH=7.4 phosphate buffered solution, utilize the luminous intensity of the probe that fluorescence detector records with the situation of change of sodium hypochlorite concentration.
Wherein horizontal ordinate is wavelength, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response.
Fig. 3 is embodiment 2,1 * 10
-5the Ru-PTZ of mol/L and sodium hypochlorite are in the 0.1mol/L of pH=7.4 phosphate buffered solution, and the luminous intensity of utilizing the probe that fluorescence detector records strengthens with the increase of sodium hypochlorite concentration, and reaches gradually the situation of balance.
Wherein horizontal ordinate is add of the concentration of sodium hypochlorite, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response.
Fig. 4 is embodiment 2,1 * 10
-5the Ru-PTZ of mol/L and sodium hypochlorite in the 0.1mol/L of pH=7.4 phosphate buffered solution, the linear relationship situation that the luminous intensity of utilizing the probe that fluorescence detector records strengthens with the increase of sodium hypochlorite concentration.
Wherein horizontal ordinate is add of the logarithm of sodium hypochlorite concentration, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe after to sodium hypochlorite concentration-response.
Fig. 5 is embodiment 3, verifies 1 * 10
-5the Ru-PTZ of mol/L, in the 0.1mol/L of pH=7.4 phosphate buffered solution, illustrates the high selectivity of sodium hypochlorite.
Wherein horizontal ordinate is the detected component adding in detection system, ordinate is the difference that adds the luminous intensity of probe after detected component to deduct to add the luminous intensity of probe before detected component, and the luminous intensity of potpourri deducts the difference of the luminous intensity of Ru-PTZ blank solution.
Fig. 6 is embodiment 4,1 * 10
-5the Ru-PTZ of mol/L and sodium hypochlorite, in the 0.02mol/L of pH=7.4 phosphate buffered solution, utilize the luminous intensity of the probe that fluorescence detector records with the situation of change of sodium hypochlorite concentration.
Wherein horizontal ordinate is wavelength, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response.
Fig. 7 is embodiment 4,1 * 10
-5the Ru-PTZ of mol/L and sodium hypochlorite are in the 0.02mol/L of pH=7.4 phosphate buffered solution, and the luminous intensity of utilizing the probe that fluorescence detector records strengthens with the increase of sodium hypochlorite concentration, and reaches gradually the situation of balance.
Wherein horizontal ordinate is the concentration of added sodium hypochlorite, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response.
Fig. 8 is embodiment 5,1 * 10
-5the Ru-A-PTZ of mol/L and sodium hypochlorite, in the 0.1mol/L of pH=7.4 phosphate buffered solution, utilize the luminous intensity of the probe that fluorescence detector records with the situation of change of sodium hypochlorite concentration.
Wherein horizontal ordinate is wavelength, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response
Fig. 9 is embodiment 5,1 * 10
-5the Ru-A-PTZ of mol/L and sodium hypochlorite are in the 0.1mol/L of pH=7.4 phosphate buffered solution, and the luminous intensity of utilizing the probe that fluorescence detector records strengthens with the increase of sodium hypochlorite concentration, reaches gradually the situation of balance.
Wherein horizontal ordinate is the concentration of added sodium hypochlorite, and ordinate is for adding after sodium hypochlorite, the luminous intensity values of probe to its response.
Figure 10 is embodiment 5,1 * 10
-5the Ru-A-PTZ of mol/L and sodium hypochlorite in the 0.1mol/L of pH=7.4 phosphate buffered solution, the linear relationship situation that the luminous intensity of utilizing the probe that fluorescence detector records strengthens with the increase of sodium hypochlorite concentration.
Wherein horizontal ordinate is the logarithm of add of sodium hypochlorite concentration, ordinate is for adding after sodium hypochlorite, probe deducts the luminous intensity values that adds probe blank solution before sodium hypochlorite to the luminous intensity values after sodium hypochlorite response, the luminous intensity of mixed solution deducts the difference of the luminous intensity of probe blank solution.
Figure 11 is embodiment 6,1 * 10
-5the Ru-PTZ of mol/L, 1 * 10
-4the sodium hypochlorite system of mol/L, and sulfuretted hydrogen is in the 0.1mol/L of pH=7.4 phosphate buffered solution, utilizes the luminous intensity of the probe that fluorescence detector records with the situation of change of concentration of hydrogen sulfide.
Wherein horizontal ordinate is wavelength, and ordinate is for adding after sulfuretted hydrogen, the luminous intensity values of probe to its response.
Figure 12 is embodiment 6,1 * 10
-5the Ru-PTZ of mol/L, 1 * 10
-4the sodium hypochlorite system of mol/L, and sulfuretted hydrogen is in the 0.1mol/L of pH=7.4 phosphate buffered solution, the luminous intensity of utilizing the probe that fluorescence detector records reduces with the increase of concentration of hydrogen sulfide, and reaches gradually the situation of balance.
Wherein horizontal ordinate is added sulfuretted hydrogen sample concentration, and ordinate is for adding after sulfuretted hydrogen, the luminous intensity values of probe to its response.
Figure 13 is embodiment 6,1 * 10
-5the Ru-PTZ of mol/L, 1 * 10
-4the sodium hypochlorite system of mol/L, and sulfuretted hydrogen is in the 0.1mol/L of pH=7.4 phosphate buffered solution, the linear relationship situation that the luminous intensity of utilizing the probe that fluorescence detector records reduces with the increase of concentration of hydrogen sulfide.
Wherein horizontal ordinate is add of the logarithm of concentration of hydrogen sulfide, and ordinate is for adding after sulfuretted hydrogen, the luminous intensity values after probe responds sulfuretted hydrogen.
Figure 14 is embodiment 7, verifies 1 * 10
-5the Ru-PTZ of mol/L, 1 * 10
-4the sodium hypochlorite system of mol/L, in the 0.1mol/L of pH=7.4 phosphate buffered solution, illustrates sulfuretted hydrogen high selectivity.
Wherein horizontal ordinate is the detected component to adding in detection system, and ordinate is the luminous intensity values that adds probe after detected component.
Figure 15 is embodiment 8, investigates 1 * 10
-5the Ru-PTZ of mol/L, in the 0.1mol/L of pH=7.4 phosphate buffered solution, illustrates the reversible cycle response of sodium hypochlorite and sulfuretted hydrogen.
Wherein horizontal ordinate is cycle index, and ordinate is for adding after sodium hypochlorite or sulfuretted hydrogen, and probe is to its luminous intensity response.
Figure 16 is embodiment 11, utilizes 1 * 10
-5the 0.1mol/L phosphate buffered solution of the Ru-PTZ(pH=7.4 of mol/L), the luminescence imaging situation of monitoring cell internal probe to sodium hypochlorite response.
Wherein (a) is that cell is 1 * 10
-5in the Ru-PTZ probe of mol/L, hatch after 150min, with 450nm wavelength, excite, the luminescence imaging situation receiving at 470nm-700nm transmitting boundary; (b) be that cell is 1 * 10
-5in the Ru-PTZ probe of mol/L, hatch the light field imaging after 150min; (c) be figure (a) and the result (b) mutually superposeing.(d) be that cell is 1 * 10
-5in the Ru-PTZ probe of mol/L, hatch after 120min, then add a small amount of phorbol 12-myristinate-13-acetic acid vinegar (PMA), continue to cultivate after 30min, with 450nm wavelength, excite the luminescence imaging situation receiving at 470nm-700nm transmitting boundary; (e) be the light field imaging corresponding with (d); (f) be figure (d) and the result (e) mutually superposeing.
Figure 17 is embodiment 12, utilizes 1 * 10
-5the Ru-PTZ of mol/L is in the 0.1mol/L of pH=7.4 phosphate buffered solution, and monitoring sodium hypochlorite and sulfuretted hydrogen are in small white mouse luminescence imaging situation during redox cycle in vivo.
Wherein (a) is to small white mouse shank cortex injection 1 * 10
-5luminescence imaging situation after the Ru-PTZ of mol/L; (b) be to inject 1 * 10 to above-mentioned small white mouse shank cortex
-5the position of the Ru-PTZ of mol/L, reinjects 1 * 10
-4the ClO of mol/L
-after luminescence imaging situation; (c) be to inject 1 * 10 to above-mentioned small white mouse shank cortex
-5the Ru-PTZ of mol/L and 1 * 10
-4the ClO of mol/L
-position, re-inject 1 * 10
-4mol/L H
2luminescence imaging situation after S; (d) be to inject H to above-mentioned small white mouse shank cortex
2the position of S, re-injects 1 * 10
-4mol/L ClO
-after luminescence imaging situation.
Table 1 is embodiment 9, utilizes 1 * 10
-5the Ru-PTZ of mol/L in the 0.1mol/L of pH=7.4 phosphate buffered solution, the measurement result to the variable concentrations sodium hypochlorite recovery.
Table 2 is embodiment 10, utilizes 1 * 10
-5the Ru-PTZ of mol/L, 1 * 10
-4mol/L sodium hypochlorite system in the 0.1mol/L of pH=7.4 phosphate buffered solution, the measurement result to the variable concentrations sulfuretted hydrogen recovery.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
In following embodiment, Ru-PTZ used is prepared as follows:
Sun Shiguo, Sun Licheng, Yang Yang ，Peng filial piety army, Fan Jiangli, Liu Fengyu.In molecule, with the bipyridyl ruthenium/osmium ECL label of phenothiazine power supplying groups, prepared by 1 record method of Chinese invention patent (publication number 101531683A) embodiment.
Embodiment 1
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In detection system, add same concentrations (1 * 10
-4mol/L) (active oxygen species adding is different types of active oxygen species: sodium hypochlorite, sodium molybdate, H
2o
2,
.oH), investigate the response condition of probe to different activities oxygen species.As can be seen from Figure 1, compare other active oxygen species, probe has single-minded, highly sensitive response to sodium hypochlorite, and responds very fast.
Embodiment 2
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.To the sodium hypochlorite that adds variable concentrations in detection system, observe its impact on probe luminous intensity.By Fig. 2, can observe, in detection system, add after sodium hypochlorite, the luminous intensity of probe evenly increases with sodium hypochlorite concentration, simultaneously known in conjunction with Fig. 3, when the concentration of sodium hypochlorite reaches 1 * 10
-4during mol/L, the luminous intensity increase of probe reaches capacity, and after this luminous intensity of probe no longer changes with the increase of sodium hypochlorite concentration.Known according to Fig. 4, the luminous intensity values (L of probe
max) with the logarithm (log[ClO of sodium hypochlorite concentration
-]) 1 * 10
-9mol/L～1 * 10
-4in mol/L sodium hypochlorite concentration range, present good linear relationship, linear equation is: L
max=546.28+54.38log[ClO
-], linearly dependent coefficient is 0.9961.According to formula, calculate (LOD=3 σ/k, in formula: σ represents that sodium hypochlorite adds the standard deviation of probe luminous intensity before, k represents the slope of linear relationship between probe luminous intensity and sodium hypochlorite concentration logarithm) known, the detection of sodium hypochlorite is limited to 1.8 * 10
-11mol/L.
Embodiment 3
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In detection system, add variety classes, 1 * 10
-4the interfering ion of mol/L or material, and then add 1 * 10
-4whether the sodium hypochlorite of mol/L, investigates the interfering ion or the material that add, can exert an influence to detecting, thereby investigate the selectivity of probe to sodium hypochlorite.Known according to Fig. 5, probe to the response of added several interfering ions or material a little less than, and sodium hypochlorite is had to very strong response.Even if so the in the situation that of above-mentioned interference component or material existence, probe still can produce selectivity, high-sensitive response to sodium hypochlorite.
Embodiment 4
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.02mol/L that the buffer solution using is pH=7.4.To the sodium hypochlorite that adds variable concentrations in detection system, observe its impact on probe luminous intensity.By Fig. 6, can see, when adding after sodium hypochlorite in detection system, the luminous intensity of probe evenly increases with sodium hypochlorite concentration, simultaneously known in conjunction with Fig. 7, when the concentration of sodium hypochlorite reaches 1 * 10
-4during mol/L, the luminous intensity increase of probe reaches capacity, and after this luminous intensity of probe no longer changes with the increase of sodium hypochlorite concentration.
Use LS55 type fluorescence detector, selecting the concentration of Ru-A-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.To the sodium hypochlorite that adds variable concentrations in detection system, observe its impact on probe luminous intensity.By Fig. 8, can observe, when adding after sodium hypochlorite in detection system, the luminous intensity of probe evenly increases with sodium hypochlorite concentration, simultaneously known in conjunction with Fig. 9, when sodium hypochlorite concentration reaches 1 * 10
-4during mol/L, the luminous intensity increase of probe reaches capacity, and after this luminous intensity of probe no longer changes with the increase of sodium hypochlorite concentration.Known according to Figure 10, add probe luminous intensity after sodium hypochlorite and difference (the Δ L that adds probe luminous intensity before sodium hypochlorite
max) with the logarithm (log[ClO of sodium hypochlorite concentration
-]) 1 * 10
-9mol/L～1 * 10
-6in mol/L sodium hypochlorite concentration range, present good linear relationship, linear equation is: Δ L
max=54.3315+3.15321log[ClO
-], linearly dependent coefficient is 0.99298.According to formula, calculate (LOD=3 σ/k, in formula: σ represents that sodium hypochlorite adds the standard deviation of probe luminous intensity before, k represents the slope of linear relationship between probe luminous intensity and sodium hypochlorite concentration logarithm) known, the detection of sodium hypochlorite is limited to 1.01 * 10
-9mol/L.
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.According to the experimental data of embodiment 2, first in system, add 1 * 10
-4the sodium hypochlorite of mol/L, makes the luminous intensity of probe reach maximal value, then to the sulfuretted hydrogen that adds again variable concentrations in system, observes and adds after sulfuretted hydrogen, probe luminous intensity variations situation.By Figure 11, can see, when adding after sulfuretted hydrogen in detection system, the luminous intensity of probe evenly reduces with the increase of concentration of hydrogen sulfide.Simultaneously known in conjunction with Figure 12, when the concentration of sulfuretted hydrogen sample reaches 1 * 10
-4during mol/L, the luminous intensity of probe drops to minimum, and after this luminous intensity of probe no longer changes with the increase of sulfuretted hydrogen sample concentration.Simultaneously known in conjunction with Figure 13, the luminous intensity values (L of probe
max) with the logarithm value (log[H of sulfuretted hydrogen sample concentration
2s]) 1 * 10
-9mol/L～1 * 10
-4within the scope of mol/L concentration of hydrogen sulfide, present good linear relationship, linear equation is: L
max=231.9194-62.6113log[H
2s], linearly dependent coefficient is 0.99308.According to formula, calculate (LOD=3 σ/k, in formula: σ represents that sulfuretted hydrogen adds the standard deviation of probe luminous intensity before, and k represents the slope of linear relationship between probe luminous intensity and concentration of hydrogen sulfide logarithm) known, the detection of sulfuretted hydrogen is limited to 1.2 * 10
-11mol/L.
Embodiment 7
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In order to investigate Ru-PTZ, the selectivity of sodium hypochlorite system to sulfuretted hydrogen sample, in detection system, add different types of reducing substances, observe Ru-PTZ, the response condition of sodium hypochlorite system to sulfuretted hydrogen and other reducing substances.According to Figure 14, can see, when adding different types of reducing substances in system, Ru-PTZ, sodium hypochlorite system have the selectivity of height to sulfuretted hydrogen, and response time while is shorter, can realize rapidly the detection to sulfuretted hydrogen sample.
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In order to investigate the round robin performance of Ru-PTZ luminescence probe to sodium hypochlorite and sulfuretted hydrogen, in system, add sodium hypochlorite and sulfuretted hydrogen sample respectively, add after sodium hypochlorite, make the luminous intensity values of probe reach maximum, and then be enough to make luminous intensity to get back to the sulfuretted hydrogen sample of original value to adding in system, so repeatedly.Result as shown in figure 15, draws according to Figure 15, and this probe can carry out cycle detection to sodium hypochlorite and sulfuretted hydrogen sample, and cycle index is more than 10 times.
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In order to investigate, utilize Ru-PTZ as luminescence probe, to detect the accuracy of sodium hypochlorite, the liquor natrii hypochloritis who prepares respectively 4 kinds of variable concentrations: 5.0 * 10
-9mol/L; 1.0 * 10
-7mol/L; 5.0 * 10
-6mol/L; 1.0 * 10
-4mol/L, and utilize 1 * 10
-5the Ru-PTZ of mol/L carries out respectively luminous detection to the liquor natrii hypochloritis of above-mentioned 4 kinds of variable concentrations, and each concentration detects respectively three times.Probe each time, in the linear equation of response luminous intensity values substitution embodiment 2 gained of sodium hypochlorite, is obtained to liquor natrii hypochloritis's to be measured respective concentration.Using the mean value of three detection gained response concentration as detectable concentration, calculate recovery rate and relative standard deviation (RSD).Result is as shown in table 1.Known by data analysis in table 1, while utilizing this probe in detecting sodium hypochlorite, the recovery fluctuates within the scope of 98.7%-111%.Relative standard deviation is no more than 1.54%.It is very reliable that these results all show to utilize Ru-PTZ as luminescence probe, sodium hypochlorite to be detected.
Table 1 luminescence probe Ru-PTZ(10 μ M, pH=7.4,0.1M PBS) detection sodium hypochlorite recovery result
Use LS55 type fluorescence detector, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, setting excitation wavelength is 450nm, scanning wavelength scope is 470nm-700nm, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.In order to investigate the accuracy of utilizing Ru-PTZ, sodium hypochlorite system to detect sulfuretted hydrogen, prepare respectively the hydrogen sulfide solution of 4 variable concentrations: 1.0 * 10
-8mol/L; 1.0 * 10
-7mol/L; 1.0 * 10
-6mol/L; 1.0 * 10
-4mol/L, and utilize 1 * 10
-5the Ru-PTZ of mol/L, 1.0 * 10
-4the sodium hypochlorite system of mol/L is carried out respectively luminous detection to the hydrogen sulfide solution of above-mentioned 4 kinds of variable concentrations, and each concentration detects respectively three times.By probe each time in the linear equation of response luminous intensity values substitution embodiment 6 gained of sulfuretted hydrogen, the detectable concentration meeting with a response.Using the mean value of three detection gained response concentration as detectable concentration, calculate recovery rate and relative standard deviation (RSD).Result is as shown in table 2.While utilizing this system to detect sulfuretted hydrogen, the recovery fluctuates within the scope of 102%-105%.Relative standard deviation is no more than 5.8%.It is very reliable that these results all show to utilize Ru-PTZ, sodium hypochlorite system to carry out luminous detection to sulfuretted hydrogen.
The oxidation state of table 2 luminescence probe Ru-PTZ (10 μ M Ru-PTZ+1.0 * 10
-4mol/L sodium hypochlorite, pH=7.4,0.1M PBS) detection sulfuretted hydrogen recovery result
Embodiment 11
Utilize the 0.1mol/L phosphate buffered solution of pH=7.4, prepare 1 * 10
-5mol/L Ru-PTZ luminescence probe solution.Measure 40 μ L probe solutions and join the MCF-7(human breast cancer cell with 2mL nutrient culture media) in Tissue Culture Dish, at 37 ℃, 5%CO
2cell culture incubator in hatch 150min.Then (TCS-SP2, Germany) observation of cell form on laser confocal scanning microscope.Under the same terms, the probe solution of getting 40 μ L is added in another one Tissue Culture Dish, adds a small amount of phorbol 12-myristinate-13-acetic acid vinegar (PMA) after cultivating 120min, then cultivates after 30min observation of cell form under laser confocal scanning microscope.Choose representative area, with 450nm wavelength, excite, select 470nm-700nm receiving cable to receive utilizing emitted light.As Figure 16 (a), (b), (c) shown in, when only when injecting Ru-PTZ in cell, owing to there is no hypochlorous acid, probe presents weak luminous intensity in cell.As Figure 16 (d), (e), (f) shown in, under the stimulation of PMA, produce hypochlorous acid in cell, the hypochlorous acid of generation makes the luminous obvious enhancing of probe.
Embodiment 12
Use NightOWL II LB 983 whole body optical imaging systems, selecting the concentration of Ru-PTZ luminescence probe molecule is 1 * 10
-5mol/L, the phosphate buffered solution of the 0.1mol/L that the buffer solution using is pH=7.4.Excitation wavelength is 450nm, uses the optical filter of 600-620nm to receive utilizing emitted light.In order to investigate probe for the redox response condition of sodium hypochlorite and the sulfuretted hydrogen of live body inside, select small white mouse live body to carry out luminescence imaging experiment.First, to small white mouse shank cortex injection 1mL1 * 10
-5mol/L Ru-PTZ, its luminescence imaging as shown in Figure 17 (a) shows, can find out from Figure 17 (a), independent 1 * 10
-5mol/L Ru-PTZ shows more weak luminous intensity.Then, then to the identical position of small white mouse shank cortex inject 1.0 * 10
-4the sodium hypochlorite of mol/L, luminescence imaging is as shown in Figure 17 (b).From Figure 17 (b), can find out, when to injecting 1 * 10
-5the position of mol/L Ru-PTZ, injects after sodium hypochlorite again, and luminous intensity obviously strengthens.Secondly, then to the identical position of above-mentioned small white mouse shank cortex inject 1.0 * 10
-4the sulfuretted hydrogen of mol/L, luminescence imaging is as shown in Figure 17 (c).From Figure 17 (c), can find out, when to injecting 1 * 10
-5mol/L Ru-PTZ and 1.0 * 10
-4the position of mol/L sodium hypochlorite, after inject hydrogen sulfide, the luminous intensity of probe has returned to original level again.Finally, again to above-mentioned small white mouse shank cortex same position injection 1.0 * 10
-4the sodium hypochlorite of mol/L, its luminescence imaging is as shown in Figure 17 (d).From Figure 17 (d), can see, although after injection hydrogen persulfide, the luminous intensity of probe has returned to original level, when probe runs into after sodium hypochlorite again, its luminous intensity significantly strengthens again.Above-mentioned experimental result shows, this probe can be applied to monitor the redox cycle process of the inner sodium hypochlorite of small white mouse live body and sulfuretted hydrogen.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.In embodiments of the invention, adopt phosphate buffer, other buffer systems, under suitable pH value, utilize above-mentioned luminescence probe molecule also can reach similar detection effect, also should belong to protection scope of the present invention.Several interior luminescence probe molecules with phenothiazine power supplying groups of molecule that utilize in embodiments of the invention, have only been enumerated; by luminescence analysis, detect the method for hypochlorite and sulfuretted hydrogen; luminescence probe molecule with phenothiazine power supplying groups in the molecule of other kinds also can be applied in the detection of hypochlorite and sulfuretted hydrogen under suitable condition, all belongs to protection scope of the present invention.
Claims (6)
1. a method for reversible detection hypochlorite and sulfuretted hydrogen, comprises the steps:
1. known hypochlorite concentration standard sample and luminescence probe molecule are dissolved in buffer solution, measure system luminous intensity, set up hypochlorite concentration and luminescence probe mulecular luminescence strength relationship;
2. known concentration of hydrogen sulfide standard model is added step 1. in the oxidized luminescence probe molecule buffer solution of gained, measure system luminous intensity, set up concentration of hydrogen sulfide and luminescence probe mulecular luminescence strength relationship;
3. hypochlorite sample to be measured and luminescence probe molecule are dissolved in buffer solution, measure system luminous intensity, according to step 1. gained hypochlorite concentration and system luminous intensity relationship, determine the concentration of hypochlorite in hypochlorite sample to be measured;
4. sulfuretted hydrogen sample to be measured is added step 3. in gained solution, measures system luminous intensity, according to step 2. gained concentration of hydrogen sulfide and system luminous intensity relationship, determine the concentration of sulfuretted hydrogen in sulfuretted hydrogen sample to be measured,
Wherein, described luminescence probe molecule is the metal complex with general formula I structure:
(L
1L
2)M-L
3Y
3
I
In formula I, M is Ru or Os;
Y is halide ion, ClO
4 -, BF
4 -, PF
6 -or OTs
-;
L
1and L
2be selected from independently of one another following part:
Wherein, R
1and R
2be selected from independently of one another H, C
1-18alkyl, CHO, COOH, NH
2, C
1-6alkyl amino, OH, SH, C
1-6alkoxy, C
1-6amide group, C
1-6alkyl replaces or unsubstituted benzyl, halogen or C
1-6haloalkyl;
L
3be selected from following part:
Wherein, R
3for H, CH
3or R
4;
R
4be selected from and there is general formula PTZ
1, PTZ
2or PTZ
3group,
In formula: n=1～18, m=0～18
2. method according to claim 1, is characterized in that: described L
1and L
2be selected from independently of one another following part:
Wherein, R
1and R
2be selected from independently of one another H and C
1-6alkyl.
3. method according to claim 1 and 2, is characterized in that: described L
3be selected from following part:
Wherein, R
3for H or CH
3; R
4be selected from and there is general formula PTZ
2or PTZ
3group, in formula, m=0, n=2～6.
4. method according to claim 1, is characterized in that: described luminescence probe molecule is selected from a kind of in following metal complex:
5. method according to claim 1, is characterized in that: described luminescence probe mulecular luminescence intensity is determined as follows: solution is carried out to luminescent spectrum scanning, and excitation wavelength is 450nm, and emission wavelength sweep limit is 470nm～700nm.
6. method according to claim 1, is characterized in that: the concentration of described luminescence probe molecule in buffer solution is 1 * 10
-5mol/L.
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| CN110078772A (en) * | 2019-06-20 | 2019-08-02 | 福州大学 | Fluorescence probe based on complex of iridium and preparation method thereof and the application in hypochlorous acid detects |
| CN110684525A (en) * | 2019-11-12 | 2020-01-14 | 中国科学院新疆理化技术研究所 | Colorimetric-fluorescent probe based on aggregation-induced emission effect and preparation method and application thereof |
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